Metabolic engineering of the l-serine biosynthetic pathway improves glutathione production in Saccharomyces cerevisiae

Background Glutathione is a valuable tri-peptide that is industrially produced by fermentation using the yeast Saccharomyces cerevisiae, and is widely used in the pharmaceutical, food, and cosmetic industries. It has been reported that addition of l-serine (l-Ser) is effective at increasing the intracellular glutathione content because l-Ser is the common precursor of l-cysteine (l-Cys) and glycine (Gly) which are substrates for glutathione biosynthesis. Therefore, we tried to enhance the l-Ser biosynthetic pathway in S. cerevisiae for improved glutathione production. Results The volumetric glutathione production of recombinant strains individually overexpressing SER2, SER1, SER3, and SER33 involved in l-Ser biosynthesis at 48 h cultivation was increased 1.3, 1.4, 1.9, and 1.9-fold, respectively, compared with that of the host GCI strain, which overexpresses genes involved in glutathione biosynthesis. We further examined simultaneous overexpression of SHM2 and/or CYS4 genes involved in Gly and l-Cys biosynthesis, respectively, using recombinant GCI strain overexpressing SER3 and SER33 as hosts. As a result, GCI overexpressing SER3, SHM2, and CYS4 showed the highest volumetric glutathione production (64.0 ± 4.9 mg/L) at 48 h cultivation, and this value is about 2.5-fold higher than that of the control strain. Conclusions This study first revealed that engineering of l-Ser and Gly biosynthetic pathway are useful strategies for fermentative glutathione production by S. cerevisiase. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-022-01880-8.


Background
Glutathione is the most abundant non-protein thiol compound in all living organisms [1]. Because of its important physiological functions including its ability to act as an antioxidant, a detoxifier of xenobiotics, and an immune booster [2][3][4][5][6][7][8], glutathione has been widely used in the medical, food and cosmetic industries [9,10]. Therefore, the demand for glutathione has increased in recent years.
Some studies have reported that supplying several types of amino acids during glutathione fermentation increased glutathione production in Saccharomyces

Open Access
Microbial Cell Factories *Correspondence: akondo@kobe-u.ac.jp 1 Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, Hyogo 657-8501, Japan Full list of author information is available at the end of the article cerevisiae [16,17]. The addition of l-Cys was especially effective at increasing glutathione production, and metabolic engineering of l-Cys biosynthesis was also effective in enhancing glutathione production [11]. It was also reported that addition of l-serine (l-Ser) was effective in increasing the intracellular glutathione content [17] because l-Ser is the common precursor of l-Cys and Gly (Fig. 1). This fact suggests that intracellular l-Ser supplementation by genetic engineering should improve glutathione production without external addition of l-Ser. As shown in Fig. 1, l-Ser biosynthesis is carried out by three consecutive reactions from 3-phospho-glycerate (3PG), which is an intermediate of glycolytic pathway [18].
In this study, we enhanced glutathione productivity by metabolic engineering the l-Ser biosynthetic pathway from 3PG using the GCI strain as a host. Furthermore, we attempted to combine engineering of the l-Cys and Gly biosynthetic pathways with the l-Ser biosynthetic pathway to further enhance glutathione production.

Results
Effect of external l-Ser supplementation on glutathione production by GCI strain l-Ser is a common precursor of both l-Cys and Gly, which are both substrates for glutathione biosynthesis (Fig. 1). To confirm the effect of external l-Ser supplementation on glutathione production in the GCI host strain, we measured biomass concentration (g/L), intracellular glutathione content (%), and volumetric glutathione production (mg/L) every 24 h for 72 h of fermentation with and without 300 mg/L l-Ser addition (Fig. 2). As shown in Fig. 2A, the biomass concentration was increased by addition of l-Ser (1.65 ± 0.05 g/L at 72 h) compared with no l-Ser addition (1.42 ± 0.03 g/L at 72 h). Furthermore, as shown in Fig. 2b, addition of l-Ser also increased the intracellular glutathione content (2.2 ± 0.1% at 72 h) compared with no l-Ser addition (1.5 ± 0.1% at 72 h) by about 1.5-fold. Because of the synergetic effect of the increased biomass concentration and intracellular glutathione content, the volumetric glutathione production was consistent for more than 72 h with l-Ser addition, whereas it was decreased when no l-Ser was added (Fig. 2C). However, the maximum yield of glutathione production was not enhanced by l-Ser addition at 24 h.

Glutathione production by GCI/SER3 and GCI/SER33 strains engineered in Gly and/or l-Cys biosynthetic pathways
Enhancing l-Cys and Gly biosynthesis from l-Ser with enhancing l-Ser biosynthesis would be expected to further improve glutathione production by S. cerevisiae. l-Cys and a part of Gly are synthesized from Glutathione production by GCI strains overexpressing l-Ser biosynthetic genes. A Biomass concentration (g/L). B Intracellular glutathione content (%). C Volumetric glutathione production (mg/L). D GSSG ratio (%). Blue and red filled bars represent the values after growth for 24 and 48 h, respectively. The GSSG ratio was defined as the ratio of the g-GSSG in cells to the g-total glutathione (GSH and GSSG) in cells. Values are the mean, and error bars show the standard deviation (n = 3) l-Ser through reactions catalyzed by cystathionine γ-synthase (Cys4) and cytosolic serine hydroxymethyltransferase (Shm2), respectively ( Fig. 1). In l-Ser biosynthetic genes, overexpression of SER3 or SER33 was effective to improve volumetric glutathione production (Fig. 3). Therefore, we constructed SHM2 and/ or CYS4 overexpressing strains using GCI/Vector, GCI/SER3, and GCI/SER33 as platform strains (Fig. 4). Unlike the case of the glutathione production by l-Ser biosynthetic gene overexpressing strains, the results of the strains overexpressing SHM2 and/or CYS4 using GCI/Vector, GCI/SER3, and GCI/SER33 as platform strains were complicated. The biomass concentration, intracellular glutathione content, and volumetric glutathione production of GCI/SHM2, GCI/CYS4, and GCI/SHM2/CYS4 at 48 h were apparently higher than those of the GCI/Vector ( Fig. 4A-C; Additional file 1: Table S4). On the other hand, the GSSG ratio of the GCI/SHM2 (29.6 ± 1.7%) and GCI/CYS4 (30.4 ± 2.4%) at 48 h were somewhat higher than the that of the GCI/Vector (Fig. 4D), and the GSSG ratio of the GCI/SHM2/CYS4 (26.1 ± 2.5%) at 48 h was almost same to the that of the GCI/Vector. These results indicate that overexpression of the SHM2 and/ or CYS4 is effective for glutathione production by S. cerevisiae even when l-Ser biosynthetic pathway of the platform strain was not engineered. The intracellular glutathione content and volumetric glutathione production of the GCI/SER3/SHM2, GCI/SER3/CYS4, and GCI/SER3/SHM2/CYS4 at 48 h were also higher than those of the platform strain GCI/SER3 (Fig. 4B, C; Additional file 1: Table S4). However, the biomass concentration of the GCI/SER3/SHM2 (1.73 ± 0.16 g/L) and GCI/SER3/CYS4 (1.75 ± 0.06 g/L) at 48 h were slightly higher than that of the GCI/SER3 (Fig. 4A), and that of the GCI/SER3/SHM2/CYS4 (1.58 ± 0.11 g/L) was apparently decreased. Unlike using the GCI/Vector as a platform, the GSSG ratio of the GCI/SER3/SHM2 (41.6 ± 0.2%), GCI/SER3/CYS4 (41.0 ± 0.4%), and GCI/SER3/SHM2/CYS4 (40.5 ± 2.0%) at 48 h were higher than that of the GCI/SER3 (Fig. 4D). The biomass concentration, intracellular glutathione content, and volumetric glutathione production of the GCI/SER33/SHM2 and GCI/SER33/CYS4 at 48 h were obviously decreased compared to those of the platform strain GCI/SER33, whereas those of the GCI/SER33/SHM2/CYS4 were higher than those of the GCI/SER33 (Fig. 4A-C; Additional file 1: Table S4). Meanwhile, the GSSG ratio of the GCI/SER33/SHM2 (25.8 ± 1.6%), GCI/SER33/CYS4 (27.0 ± 0.7%), and GCI/SER33/SHM2/CYS4 (27.9 ± 0.4%) at 48 h were slightly higher than that of the GCI/SER3 (Fig. 4D).

Discussion
l-Ser biosynthesis from 3PG in S. cerevisiae occurs via three consecutive reactions catalyzed by SER2, SER1, and SER3/SER33. The intracellular glutathione content of the recombinant strains individually overexpressing SER2, SER1, SER3, and SER33 were 1.4-to 1.8-fold higher than that of the control strain at 48 h (Fig. 3B). Albers et al. showed that transcription levels of SER2, SER3, and SER33 were less than that of SER1 [18]. The result that overexpression of SER2, SER3, and SER33 increased intracellular glutathione content compared with that of SER1 is likely primarily influenced by transcription levels of each gene. Furthermore, the result that the overexpression of SER3 and SER33 were the most effective to enhance the glutathione production (Fig. 3B, C) suggested that SER3 and SER33 were the most critical step in the l-Ser biosynthetic pathway, and this fact is consistent with the previous study which reported deletion of SER3 and SER33 equally influenced metabolite production [18]. Thus, we selected the GCI/SER3 and GCI/SER33 strains as the platform strain for further improvement of glutathione biosynthesis. However, simultaneous overexpression of SER3 and SER33 resulted in decreased intracellular glutathione content and volumetric glutathione production compared to those of the GCI/SER3 and GCI/SER33 strains (Fig. 3B, C). Furthermore, simultaneous overexpression of SER2, SER1, SER3, and SER33 lead to poor growth and consecutive low glutathione production ( Fig. 3A-C). These results may imply that surplus l-Ser production or decrease of 3PG by enhanced l-Ser biosynthesis exerts unfavorable effects on growth and glutathione production of host cells. The increase of GSSG ratio in the GCI/SERs strain also showed that the strain was under strong stress because GSSG ratio showed index of oxidative stress of cells [12][13][14]. However, the strains overexpressing SER3 also showed high GSSG ratio compared with the control strain. In some cases, production of GSSG help to avoid a negative feedback regulation by Gsh1, and consequently increase total glutathione production [12][13][14]. The overexpression of SER3 may lead modest stress of the cells for increased glutathione production. On the other hand, the strains overexpressing SER33 showed slightly lower GSSG ratio than that of the control strains. These different effects of overexpression of SER3 and SER33 on GSSG ratios may show different mechanisms of improved glutathione production.
To further improve glutathione production by S. cerevisiae, we additionally overexpressed SHM2 and/or CYS4 genes encoding cytosolic serine hydroxymethyltransferase and cystathionine β-synthase, respectively (Fig. 1). Generally, cystathionine β-synthase encoded by CYS4 catalyzes condensation of l-Ser and l-homocysteine to produce l-cystathionine and H 2 O. Consecutively, l-cystathionine is broken down into l-Cys, 2-oxobutanoic acid, and ammonia by cystathionine γ-lyase (CYS3). However, we didn't adopt overexpression of CYS3 in this study because Cys4 of S. cerevisiae can directly catalyze l-Ser and H 2 S to l-Cys and H 2 O [19] as shown in Fig. 1.
Unexpectedly, overexpression of SHM2 and/or CYS4 effectively enhanced biomass concentration, intracellular glutathione content, and consecutive volumetric glutathione production even when the GCI strain was used as a platform.
l-Cys is known as a late-limiting precursor for glutathione biosynthesis, and therefore a lot of studies have employed external addition of l-Cys to the culture medium for enhancing glutathione production [16,17,[20][21][22][23]. Enhancing l-Cys biosynthesis with or without enhancing l-Ser biosynthesis also improved glutathione production of S. cerevisiae not only in this study, but also in previous study [11]. However, the result that enhancing Gly biosynthesis by genetic engineering on glutathione production has not been reported so far. According to the report of Wang et al., l-Glu abundantly exists in S. cerevisiae cells during fermentative glutathione production (185.9 ± 10 μmol/g-dry cell) [24] and intracellular Gly concentration (0.44 ± 0.15 μmol/g-dry cell) was almost the same as that of l-Cys (0.42 ± 0.2 μmol/g-dry cell) without addition of these precursors to the medium. In other cases, l-Cys concentration was lower than that of Gly; however, Gly concentration was much lower than that of l-Glu [25,26]. Thus, there is a possibility that enhancing Gly biosynthesis promotes glutathione biosynthesis. In fact, addition of Gly to the medium improved glutathione production in a previous study [24]. On the other hand, Alfafara et al. reported that supplementation of Gly in the medium had no effect on the glutathione production of S. cerevisiae [16]. This disagreement may be caused by various factors of fermentation such as genotype of host S. cerevisiae, preculture conditions, initial cell concentration, medium composition, and so on. Indeed, even in this study, overexpression of SHM2 in GCI/SER33, GCI/SER3/SER33, and GCI/SERs didn't improve glutathione production (Fig. 4B, C).
Among the GCI/SER3/SER33 and GCI/SERs strains overexpressing SHM2 and/or CYS4, only GCI/SER3/S ER33/SHM2/CYS4 and GCI/SERs/CYS4 showed modestly increased volumetric glutathione production compared to the platform strains (Fig. 5C). If the cause of poor glutathione production by GCI/SER3/SER33 and GCI/SERs were surplus l-Ser biosynthesis, this disadvantage could be solved by overexpression of SHM2 and/or CYS4 regardless of the combination. However, the recovery of the cell growth by overexpression of SHM2 and/ or CYS4 in GCI/SERs seemed to be the result that surplus biosynthesized l-Ser was metabolizing to Gly and l-Cys (Fig. 5A, D). However, overexpression of SHM2 and/or CYS4 had no effect or decreased cell growth when GCI/SER3/SER33 was used as a platform (Fig. 5A). These results imply that there may be exquisite and complicated balance between supplies of l-Ser, l-Cys, and Gly, in enhanced glutathione production of S. cerevisiae. In this study, GCI/SER3/SHM2/CYS4 showed the highest volumetric glutathione production at 48 h (64.0 ± 4.9 mg/L) among all strains constructed, and this yield is about 2.5-fold higher than that of the GCI/Vector strain. In other reports about fermentative glutathione production using yeasts, feeding of various precursors and carbon sources, and optimization of growth conditions have been often employed [23,[27][28][29][30]. In many cases adopting these strategies, the fermentation was carried out in nutrient rich condition with high cell density using glutathione high-producing strains and leached about 100-2500 mg/L of glutathione [31]. For example, Wen et al. reported 2190 mg/L of glutathione production in nutrient rich medium supplemented with l-Cys, l-Glu, and Gly using glutathione high-producing S. cerevisiae T65 strain under high cell density and glucose feeding conditions [28]. Comparing with glutathione production of these reports, the maximum glutathione production of this study (64.0 ± 4.9 mg/L) is very low. The S. cerevisiae strains in this study were grown in the nutrient limited minimal medium with low initial cell densities. These growth conditions may lead poor glutathione production compared with those of other reports. However, our objective of this study is to provide a new strategy that modification of l-Ser, l-Cys, and Gly biosynthetic genes improves glutathione production of S. cerevisiae. Indeed, glutathione production studies focused of genetic engineering reported about 10-300 mg/L of glutathione production [32] and the reason for these low yields may be the not optimized culture conditions. Furthermore, in many of these studies, genes directly involved in glutathione synthesis such as GSH1 [33], gshF encoding bifunctional glutathione synthase [34], and PRO1 encoding γ-glutamyl kinase [35] are overexpressed. In such situations, we focused on l-Ser biosynthetic genes and first revealed that engineering of l-Ser and Gly biosynthetic pathway are useful strategies for fermentative glutathione production by S. cerevisiase. These our findings may be applicable for other glutathione high-producing yeast strains to further improve glutathione production.
Transformation of S. cerevisiae was carried out using the lithium acetate method as described previously [38,39]. Transformants were selected by leucine (pATP405) and uracil (pATP406) auxotrophies. The insertion of the target gene in each transformant was confirmed by PCR method using appropriate primers.

Glutathione analysis
Reduced glutathione (GSH) and oxidized glutathione (GSSG) in recombinant S. cerevisiae cells were determined as described in our previous report [40]. Cell cultures (1 mL) grown for glutathione production were sampled every 24 h, and the OD 600 of the culture samples were measured using a UVmini-1240 Spectrometer (Shimadzu, Kyoto, Japan). The cells were pelleted by centrifugation (16,000×g, 1 min) and rinsed with Milli-Q water twice. The rinsed cell pellets were re-suspended in Milli-Q water and incubated at 95 °C for 3 min, then cooled immediately on ice for 3 min and the supernatant was separated by centrifugation (16,000×g, 1 min). GSH and GSSG concentrations in the supernatant were determined by HPLC (Shimadzu) equipped with a YMC-Pack ODS-A column (YMC, Kyoto, Japan). The operating conditions were 30 °C with 50 mM potassium dihydrogen phosphate buffer (pH 2.8) and 10 mM sodium 1-heptanesulfonate as the mobile phase at a flow rate of 1.0 mL/ min, and detection was performed with an ultraviolet detector SPD-20A (Shimadzu) at 210 nm. Volumetric glutathione production (mg/L-broth) and intracellular glutathione content (%) were calculated from OD 600 and used to determine glutathione concentration, as previously described [40].